Sphingosine Kinase 1 Plays an Important Role in Atorvastatin-Mediated Anti-Inflammatory Effect against Acute Lung Injury

Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor and inhibits cholesterol synthesis. Recently, atorvastatin also showed anti-inflammatory effect in acute lung injury, ameliorating pulmonary gas-blood exchanging function. Sphingosine kinase 1 plays a central role in endothelial (EC) cytoskeleton rearrangement and EC barrier integrity regulation. In this study, the role of sphingosine kinase 1 in atorvastatin anti-inflammatory effect against acute lung injury was investigated. Both wild-type (WT) and SphK1-/- mice were challenged with high tidal volume ventilation (40 ml/kg body weight, 65 breathing/min, 4 hours). The acute lung injury was evaluated and the mechanisms were explored. In WT mice, atorvastatin treatment significantly decreased acute lung injury responding to high tidal volume ventilation (HT), including protein, cellular infiltration, and cytokine releasing; comparing to WT mice, SphK1-/- mice showed significantly worsen pulmonary injuries on HT model. Moreover, the atorvastatin-mediated anti-inflammatory effect was diminished in SphK1-/- mice. To further confirm the role of SphK1 in VILI, we then compared the inflammatory response of endothelial cells that were isolated from WT and SphK1-/- mice to cyclic stretching. Similarly, atorvastatin significantly decreased cytokine generation from WT EC responding to cyclic stretching. Atorvastatin also significantly preserved endothelial junction integrity in WT EC against thrombin challenge. However, the inhibitory effect of atorvastatin on cytokine generation induced by cyclic stretching was abolished on SphK1-/- mice EC. The endothelial junction integrity effects of atorvastatin also diminished on SphK1-/- mouse EC. Signal analysis indicated that atorvastatin inhibited JNK activation induced by cyclic stretch. SphK1 knockout also blocked atorvastatin-mediated VE-cadherin junction enhancement. In summary, by inhibition of MAPK activity and maintenance of EC junction homeostasis, SphK1 plays a critical role in atorvastatin-mediated anti-inflammatory effects in both cellular and in vivo model. This study also offers an insight into mechanical stress-mediated acute lung injury and potential therapy in the future.

Erythrocytes increase endogenous sphingosine 1-phosphate (S1P) levels as an adaptive response to SARS-CoV-2 infection

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carrier serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier changing shift from SA to HDL, which probably prevented an even further drop of S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBC) were significantly increased in COVID-19 patients compared to healthy controls due to upregulation of S1P producing sphingosine kinase 1 and downregulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Impaired Sphingosine-1-Phosphate Synthesis Induces Preeclampsia by Deactivating Trophoblastic YAP (Yes-Associated Protein) Through S1PR2 (Sphingosine-1-Phosphate Receptor-2)-Induced Actin Polymerizations

Incomplete spiral artery remodeling, caused by impaired extravillous trophoblast invasion, is a fundamental pathogenic process associated with malplacentation and the development of preeclampsia. Nevertheless, the mechanisms controlling this regulation of trophoblast invasion are largely unknown. We report that sphingosine-1-phosphate synthesis and expression is abundant in healthy trophoblast, whereas in pregnancies complicated by preeclampsia the placentae are associated with reduced sphingosine-1-phosphate and lower SPHK1 (sphingosine kinase 1) expression and activity. In vivo inhibition of sphingosine kinase 1 activity during placentation in pregnant mice led to decreased placental sphingosine-1-phosphate production and defective placentation, resulting in a preeclampsia phenotype. Moreover, sphingosine-1-phosphate increased HTR8/SVneo (immortalized trophoblast cells) cell invasion in a Hippo-signaling–dependent transcriptional coactivator YAP (Yes-associated protein) dependent manner, which is activated by S1PR2 (sphingosine-1-phosphate receptor-2) and downstream RhoA/ROCK induced actin polymerization. Mutation-based YAP-5SA demonstrated that sphingosine-1-phosphate activation of YAP could be either dependent or independent of Hippo signaling. Together, these findings suggest a novel pathogenic pathway of preeclampsia via disrupted sphingosine-1-phosphate metabolism and signaling-induced, interrupted actin dynamics and YAP deactivation; this may lead to potential novel intervention targets for the prevention and management of preeclampsia.

Sphingosine Kinase 1 Signaling in Breast Cancer: A Potential Target to Tackle Breast Cancer Stem Cells

Sphingosine kinases (SPHKs) are conserved lipid enzymes that catalyze the formation of sphingosine-1-phosphate (S1P) through ATP-dependent phosphorylation of sphingosine. Two distinct SPHK isoforms, namely SPHK1 and SPHK2, have been identified to date, and the former has been implicated for its oncogenic roles in cancer development and progression. While SPHK1 signaling axis has been extensively studied in non-stem breast cancer cells, recent evidence has emerged to suggest a role of SPHK1 in regulating cancer stem cells (CSCs). With the clinical implications of CSCs in disease relapse and metastasis, it is believed that therapeutic approaches that can eradicate both non-stem cancer cells and CSCs could be a key to cancer cure. In this review, we first explore the oncogenic functions of sphingosine kinase 1 in human cancers and summarize current research findings of SPHK1 signaling with a focus on breast cancer. We also discuss the therapeutic potentials and perspectives of targeting SPHK1 signaling in breast cancer and cancer stem cells. We aim to offer new insights and inspire future studies looking further into the regulatory functions of SPHK1 in CSC-driven tumorigenesis, uncovering novel therapeutic avenues of using SPHK1-targeted therapy in the treatment of CSC-enriched refractory cancers.

High-resolution MD simulation studies to get mechanistic insights into the urea-induced denaturation of human Sphingosine kinase 1

Background: Sphingosine kinase 1 (SPhK1) is a crucial signaling enzyme involved in cell proliferation, cellular survival, stimulation of angiogenesis, and apoptosis prevention. Recently, we have reported the unfolding kinetics of SPhK1 using molecular dynamics (MD) simulation, circular dichroism and fluorescence spectroscopy. We found that SPhK1 showed a biphasic unfolding with an intermediate state (~ 4.0 M urea). Objective: We aim to understand the impact of MD simulation duration on the structure, function and dynamics of proteins. In order to get deeper insights into the folding mechanism an extended MD simulation is required. Method: Here, we extended the MD simulations time scale from 100 to 300 ns on SPhK1 at increasing urea concentration to explore structural changes in the SPhK1. Results: The results suggested a constant form of the unfolding of SPhK1 upon extending the simulation time scale at different urea concentrations. Furthermore, we showed step by step unfolding and percentage of secondary structure contents in SPhK1 under the influence of urea at each concentration. Conclusion: The results from the current work revealed a uniform pattern of the SPhK1 unfolding at different urea concentrations. This study provides deeper mechanistic insights into the urea-induced denaturation of SPhK1.

DDRE-19. SPHINGOSINE KINASE 1 AS A THERAPEUTIC TARGET FOR IDH1-R132H mut GLIOMA

BACKGROUND Our study aimed to identify vulnerabilities within sphingolipid metabolism with potential to translate to therapeutics. While the vital role of sphingolipids in maintaining rheostat balance and as secondary messengers for signaling pathways (involving proliferation, invasion, migration, and angiogenesis) has been well-documented, their role has not been widely investigated in gliomas. Therefore, metabolic analysis of sphingolipid pathway for IDH1-R132H (IDH1 mut ) glioma cell lines was conducted in order to elucidate susceptible targets. METHODS Global sphingolipid quantification utilized high-throughput LCMS analysis. Pathway protein expression was measured via Western blots in vitro and derived from patients using The Cancer Genome Atlas analysis. RESULTS We probed the impact of decreasing D-2HG on the sphingolipid metabolism after treating a panel of IDH1 mut glioma cells with IDH1-R132H mut inhibitor, AGI5198. This revealed significant downregulation of N,N-dimethylsphingosine (NDMS), C17-sphingosine, and C18-sphinganine. Coincidentally, sphingosine-1-phosphate (S1P) was significantly upregulated in these gliomas. We conducted rational drug screen which revealed that inhibition of SPHK1 with N,N-dimethylsphingosine in combination with C17-sphingosine triggered biostatic dose-response across IDH1 mut gliomas and low impact on IDH WT glioblastoma (GBM) cells. Western analysis revealed that the IDH1 mut gliomas and IDH WT GBM expressed sphingosine kinase-1 (SPHK1). Data also unveiled a discovery that SPHK2 was highly expressed in the GBM cells while remarkably absent in the glioma cells. CONCLUSION Herein, we provide evidence that certain IDH1 mut gliomas present epigenetic silencing of SPHK2 which creates dependency on SPHK1 for S1P; thus, increasing sensitivity to targeting sphingolipid metabolism, and creating susceptibility to proliferation arrest and subsequent cellular death. S1P production has been reported to be elevated particularly for malignant glioblastomas in prior studies; whereas our research revealed that it is relatively low in IDH mut by comparison with IDH WT tumor cells. These findings suggest targeting the sphingolipid metabolism may present a promising strategy to improve survival for patients diagnosed with IDH1 mut gliomas.

Sphingosine Kinase 1(SphK1) – A Novel Marker for Cancer Risk Assessment in Oral Premalignancy

Introduction Head and neck cancer is the sixth most common cancer worldwide, resulting in approximately 550,000 diagnoses of new cases and 300,000 deaths per year globally. Overexpression of Sphingosine kinase - 1 (SphK1) is found in head and neck squamous cell carcinoma (HNSCC) from early to advanced stages and is associated with tumor progression, invasion, metastasis and poor prognosis. This study assesses the expression of sphingosine kinase - 1 (SphK1) in oral leukoplakia and oral squamous cell carcinoma (OSCC) and suggests its role as a potential biomarker tool for cancer risk assessment in oral leukoplakia. Methods In this retrospective study, eighty-two (n = 82) archival formalin-fixed paraffin blocks consisting of 10 normal tongue mucosa (Group A), 42 cases of oral leukoplakia (Group B) and 30 cases of OSCCs (Group C) were selected and subjected to immunohistochemical staining for anti-rabbit SphK1 antibody. The three different groups were compared for the presence of Sphk1 expression. The clinicopathological parameters such as age, gender, habits, histopathology, and Sphk1 expression were analyzed and compared between the malignantly transformed leukoplakia with untransformed leukoplakia. Results Positive SphK1 expression was found in 18 out of 42 (42.9 %) cases of oral leukoplakia and 17 out of 30 (56.7 %) cases of OSCCs while there was no SphK1 expression in normal tongue mucosa. The expression of SphK1 among three different groups of tissue samples was statistically significant (P = 0.007). The correlation between the malignant transformed and untransformed leukoplakia lesion with respect to SphK1 expression was also found to be statistically significant (P= 0.007) Conclusions Positive SphK1 expression in oral leukoplakia is suggestive of an increased risk for malignant transformation which can be used as a biomarker tool. Higher SPHK1 expression in the OSCC may suggest an important role in the early stages of tumorigenesis.